Class V myosins (myoV) play crucial roles in actin-based organelle transport and membrane trafficking. There are three mammalian isoforms (Va, Vb, and Vc) with different tissue specificities and functions. Two of these isoforms (Va and Vb) are processive, meaning that they can take multiple steps on actin before dissociating. MyoVc is non-processive, yet all function in cargo transport. Much attention has been given to the in vitro study of single motor motion on individual actin filaments, primarily using a constitutively active truncated version of myoVa. An overarching goal of this proposal is to systematically build complexity into the study of class V myosin motors, using three complementary approaches. The first is to investigate the properties of the full-length motor, and how cargo impacts on its function. The second is to gain insight into how myoV motors work together, because cellular cargo is transported by multiple motors. The third is to introduce myoV motors into cells and track their motion, or the motion of native cargo driven by motors with known properties. In this context, we will assess differences between the three myoV isoforms. In Aim 1 we will determine how the single molecule properties of regulated full- length myoVa differ from those of constitutively active constructs, in the absence or presence of cargo. Run lengths, speed, and stepping patterns of single full-length myoVa motors, in the absence or presence of cargo, will be determined. We will test if cargo adapter proteins affect motor function beyond initial activation. In Aim 2 we investigate how two myoV motors co-ordinate their motion and force generation under unloaded and loaded conditions. DNA scaffolds that bind exactly two motors will be used to determine the effect that myoV motors have on each others' properties, under unloaded and loaded conditions. Parameters to be measured are speed, run length, step size, and stall force. Various combinations of motors will be tested. The results have implications for the behavior of multiple motors that are mechanically coupled by being bound to the same intracellular cargo. In Aim 3 we compare and contrast the motion of myoVa, myoVb, and myoVc within a living cell. The trajectories of Quantum-dot (Qdot) labeled myoV constructs, introduced into mammalian cells by pinocytosis, will be followed by high resolution total internal reflection fluorescence (TIRF) microscopy and single particle tracking. Movement of the two processive myoV isoforms (myoVa and myoVb) will be compared to non-processive myoVc. Different cell types with varying actin architectures will be used. A complementary inducible cargo trafficking assay will be used to target motor constructs to native cargo. We believe that a combined approach as presented here provides the greatest potential to move the field forward.